Endocardial endothelial (EE) dysfunction and accumulation of oxidized-matrix between endothelial and muscle are the hallmarks of congestive heart failure (CHF). The overall objective of this project is to understand the mechanism of oxidized-matrix accumulation and EE dysfunction in CHF. Previous studies have suggested an association between induction of oxidative stress, decrease in endothelial cell density, activation of matrix metalloproteinase (MMP), collagenolysis, and repression of cardiac tissue inhibitor of metalloproteinase (CIMP) in CHF. The novelty of this proposal is that among all known tissue inhibitors of metalloproteinase (TIMP), the TIMP-4 (i.e. CIMP) is highly expressed in the heart. The purpose of this proposal is to test a central hypothesis that oxidized-matrix accumulation and EE dysfunction are due to increased levels of MMP activity, and collagenolysis. These levels are associated with decreased levels of EE nitric oxide, and CIMP in response to increased levels of reactive oxygen species (ROS) during protracted cycles of ischemia/reperfusion in volume overload. The increased levels of CIMP protects EE against oxidative and proteolytic stresses. We will test the central hypothesis by the following three specific aims: L To determine whether CIMP decreases oxidative stress by increasing EE nitric oxide concentration. Plasma and left ventricle levels of nitric oxide, ROS, and nitrotyrosine will be measured in chronic volume overload arteriovenous fistula mice treated with and without CIMP. 2_ To determine whether CIMP protein transfer inhibits collagenolysis. MMP activity will be measured by in situ zymography. The levels of CIMP activity will be measured by reverse zymography. Total collagen and its degradation fragments will be measured by Western-blot analysis using anti-collagen antibody. 3-- To determine whether CIMP ameliorates endocardial endothelial dysfunction. Contractile responses to acetylcholine, bradykinin, and nitroprusside in cardiac rings will be measured in a tissue myobath. These studies will enable us to determine whether CIMP improves the hearts response to nitric oxide donors. Identification of major players involved in the control of oxidative and proteolytic stresses will help to develop strategies to prevent CHF.